The lupus anticoagulant (LA) is an immunoglobulin (IgG, IgM, or a mixture of both) which interferes with one or more of the in vitro phospholipid dependent tests of coagulation (activated partial thromboplastin time APTT!; prothrombin time PT!; dilute Russell Viper Venom Time dRVVT!). In contrast to specific inhibitors of coagulation proteins, LA has no reactivity with any of the individual coagulation factors. The name is a misnomer since the vast majority of patients do not have underlying systemic lupus erythematosus (SLE). More commonly, LA is secondary to infections, drugs (e.g. chlorpromazine, quinidine, procainamide) or it may be seen in an autoimmune disease which has recently been described: Primary Antiphospholipid Antibody Syndrome.
Paradoxically, LA is not associated with clinical bleeding unless there is some associated hemostatic defect (e.g. thrombocytopenia). Approximately 30 to 40% of patients with LA have a history of venous and arterial thromboembolic events. For a number of years, there has been much discussion as to whether LA was causative, a consequence, or coincident with thrombosis. More recent work in animal models would suggest that indeed LA is a cause of a thrombotic predisposition. other clinical manifestations of LA include recurrent fetal loss, intra-uterine fetal growth retardation, and prematurity. Also, LA may be associated with thrombocytopenia or autoimmune hemolytic anemias. Two recent excellent reviews discuss LA and its closely related antibody: anticardiolipin antibodies Triplett D. A., Brandt J. T., Lupus Anticoagulants: Misnomer, Paradox, Riddle Epiphenomenon. Hematol. Pathol. 2, 121-143, 1988; Love P. E., Santoro S. A., Antiphospholipid Antibodies: Anticardiolipin and the Lupus Anticoagulant in Systemic Lupus Erythematosus (SLE) and in Non-SLE Disorders. Ann. Int. Med. 112, 682-698, 1990!.
In most cases, LA is detected serendipitous as a result of an unexplained prolonged APTT and/or PT. Typically, an abnormal APTT is associated with quantitative or qualitative deficiencies in factors XII, XI, IX, VIII, V, or X while PT prolongation generally indicates a deficiency in either factor II, V, VII, X, or fibrinogen. Mixing patient plasma with a source of normal platelet poor plasma will result in lack of correction of the prolonged APTT and/or PT. Lack of correction is a sine qua non for the diagnosis of an inhibitor (synonym circulating anticoagulant).
The diagnosis of LA is often difficult. Commercially available APTT reagents show a wide range of sensitivity to LA and there appears to be differences betwween IgG and IgM LA. In addition to the APTT, other tests have been used to screen for LA including: dilute Russell Viper Venom Time (dRVVT), Kaolin Clotting Time, and dilute APTT. The performance of these tests is difficult requiring mixing patient and normal plasma in the case of the Kaolin Clotting Time and dilute APTT. Consequently, these tests are not readily automated with conventional coagulation instrumentation. Furthermore, if commercial freeze-dried plasmas are used as a source of normal plasma for the mixing studies, there may be false negative results due to a high content of phospholipids in the lyophilized commercial preparations.
Once a patient plasma has been established as having a prolonged screening study with lack of correction by mixing with normal platelet poor plasma, it is necessary to confirm the phospholipid specificity of the inhibitor. Two contrasting approaches have been utilized. The first of these employs a dilute phospholipid test system (e.g. tissue thromboplastin inhibition TTI!) to accentuate the inhibitor effect. The second approach utilizes a source of excess phospholipids (e.g. Platelet Neutralization Procedure PNP!) to "bypass" or "neutralize" the LA. Comparative analysis of these two different approaches suggests the PNP is more sensitive than the TTI.
In addition to the heterogeneity of commercial available reagents, patient plasmas demonstrate remarkable heterogeneity suggesting that there is a family of antibodies with LA activity. The problems of diagnosing LA have been highlighted by the deliberations of the SSC Subcommittee for Standardization of Lupus Anticoagulants (Exner, T. et al., SSC Subcommittee for the Standardization of Lupus Anticoagulants, Guidelines for Testing and Revised Criteria for Lupus Anticoagulants. Thromb. Haemost. 65, 320-322, 1991).
The venoms of several snake species contain enzymes that convert the zymogen prothrombin into the enzyme thrombin and/or its catalytically active precursor meizothrombin. Both activation products convert fibrinogen into fibrin and thereby cause plasma coagulation. Also, both thrombin and meizothrombin catalyze the hydrolytic release of chromophore from synthetic chromogenic thrombin sensitive substrates. Some of these snake venom prothrombin activators do not require a cofactor while a second group depends on the presence of calcium ions and phospholipid. A third group needs factor V in addition to calcium and phospholipid. A review on snake venom prothrombin activators is presented by: Rosing J., Tans G., Thromb. Haemost. 65, 627-630, 1991.
Phospholipid dependent prothrombin activators whose potency is enhanced by phospholipid but not by factor V have been found in the venom of snakes belonging to the Elapidae family, especially members of the Oxyuranus and Psuedonaja genera. A method for the purification of the prothrombin activator from the venom of Psuedonaja textilis using chromatography on cancanavalin A-sepharose and gel filtration is described by Masci P. P. et al., Biochemistry International 17, 825, 1988. A commercial preparation of the activator prepared according to this method is available from Venom Supplies, Tanunda, Australia. The prothrombin activator isolated from P. textilis venom according to Masci et al. is a protein with a molecular mass of greater than 200,000 daltons consisting of several non-covalently linked subunits as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). The activator according to Masci et al. (1988) was able to clot citrated plasma in the absence of calcium ions. Its plasma clotting activity was, however, stimulated 2.5 fold in the presence of calcium but no additional stimulation was observed with the addition of phospholipids.
Prothrombin activators which are insensitive to phospholipids can be isolated from venoms of snakes belonging to the family Viperidae, especially from venoms of species belonging to the genera Echis, Trimeresurus, and Bothrops using conventional protein separation techniques as described by R. K. Scopes, Protein Purification, Springer-Verlag, New York, Heidelberg, Berlin, 2nd edition, (1987). A review on the zoological classification of venomous snakes can be found in G. Underwood, Classification and distribution of venomous snakes in the world. In: C. Y. Lee (ed). Snake Venoms p. 15-40, Springer Verlag: Berlin, Heidelberg, New York (1979). A specific method for the isolation of the prothrombin activator from Bothrops atrox venom is described by Hofmann H. and Bon C., Biochemistry 26, 772 (1987) and the method for the preparation of ECARIN (phospholipid independent prothrombin activator derived from Echis carinatus venom) is provided by Morita T. and Iwanaga S., J. Biochem. 83, 559 (1978). ECARIN activator is commercially available from Pentapharm Ltd., Basle, CH. One ECARIN activator unit is the amount of ECARIN activator which under defined conditions generates one International Unit (U) of enzyme activity from prothrombin as measured with the synthetic chromogenic thrombin substrate Tos-Gly-Pro-Arg-pNA (1 U being the amount of enzyme which hydrolyzes 1 .mu.M of substrate per minute under standard conditions).